The BCHE and ACHE genes encoding the acetylcholine hydrolyzing enzymes butyrylcholinesterase (BuChE or BChE, EC 3.1.1.8) and acetylcholinesterase (AChE, EC 3.1.1.7) are expressed in muscle and nerve, hematopoietic cells, embryonic tissue and germ cells. The ACHE and BCHE genes, although significantly different from each other in nucleotide sequence, are thought to be derived from a common ancestral gene. ACHE maps to chromosome 7q22 and encodes the primary enzyme, acetylcholinesterase (AChE, E.C. 3.1.1.7), which terminates neurotransmission at synapses and neuromuscular junctions. BCHE maps to 3q26 and encodes butyrylcholinesterase (BChE, E.C. 3.1.1.8), a homologous serum esterase with somewhat broader substrate specificity.
The text Human Cholinesterases and Anticholinesterases by Soreq and Zakut (Academic Press, Inc., 1993) provides a summation of the biochemical and biological background as well as the molecular biology of human cholinesterase genes. The text in its entirety is incorporated herein by reference. Further, the text Transgenic Xenopus by Seidman and Soreq (Humana Press, 1996) provides a summation of the development of the Xenopus transgenic animal model. The text in its entirety is incorporated herein by reference. Articles by Beeri et al, 1995; Karpel et al, 1996; and the review articles by Lev-Lehman et al (1997) and Grifman et al (1995, 1997) provide further information on the development of antisense ACHE oligomers, the parameters for choosing sequences and testing for efficacy, as does co-pending U.S. patent application Ser. No. 08/318,826 assigned to the same assignee and incorporated herein by reference.
Briefly, both AChE and BuChE include the peptide motif S/T-P-X-Z, which makes them potential substrates for phosphorylation by cdc2 kinases, the general controllers of the cell cycle. Most other substrates of cdc2 kinases perform biological functions necessary for cell cycle-related processes. Thus, interference with either CHE or cdc2 transcription processes may be expected to divert and/or arrest cell division, and controlling these processes can be useful for several, medically important procedures.
Biochemical and histochemical analyses indicate that both AChE and BuChE are expressed, in high levels, in various fetal tissues of multiple eukaryotic species where cholinesterases (ChEs) are coordinately regulated with respect to cell proliferation and differentiation. The specific role to be attributed to ChEs in embryonic development may hence be related with cell division, so that their biological function(s) in these tissues are tentatively implicated in the control of organogenesis.
In addition to its presence in the membranes of mature erythrocytes, AChE is also intensively produced in developing blood cells in vivo and in vitro and its activity serves as an acceptable marker for developing mouse megakaryocytes. Furthermore, administration of acetylcholine analogues as well as cholinesterase inhibitors has been shown to induce megakaryocytopoiesis and increased platelet counts in the mouse, implicating this enzyme in the commitment and development of these hematopoietic cells.
The DNAs coding for human BuChE and AChE have been cloned and the human CHE1 locus has been mapped to the 3q26-ter chromosomal domain that is subject to aberrations in leukemias accompanied by abnormal megakaryocytopoiesis and platelet counts. Co-amplification of the ACHE and BCHE genes was subsequently observed in leukemias and platelet disorders. The hemopoietic system thus appears to be subject to developmental control as affected by the expression of the ChEs.
A major hydrophilic form of AChE with the potential to be "tailed" by non-catalytic subunits is expressed in brain and muscle whereas a hydrophobic, phosphoinositide (PI)-linked form of the enzyme is found in erythrocytes. Two sublines of the human erythroleukemic K-562 cell line were shown to express the PI-linked form of AChE, however, with different structural properties of the PI moiety.
Alternative exons encoding the C-terminal peptide in AChE were shown to provide the molecular origins for the amphiphilic (PI)-linked and the hydrophilic "tailed" form of AChE in Torpedo electric organ. The existence of corresponding alternative exons and homologous enzyme forms in mammals suggested that a similar mechanism may provide for the molecular polymorphism of human AChE. cDNAs reported to date from mammalian brain and muscle encode the hydrophilic AChE form. Nonetheless, RNA-protection and PCR analyses have demonstrated the existence of two rare alternative AChEmRNAs in mouse hemopoietic cells.
More specifically, three alternative AChE-encoding mRNAs have been described in mammals. The dominant brain and muscle AChE (AChE-T) found in the neuromuscular junction (NMJ) is encoded by an mRNA carrying exon E1 and the invariant coding exons E2, E3, and E4 spliced to alternative exon E6 [Li et al., 1991; Ben Aziz-Aloya et al., 1993]. AChEmRNA bearing exons E1-4 and alternative exon E5 encodes the glycolipid phosphatidylinositol (GPT)-linked form of AChE characteristic of vertebrate erythrocytes (AChE-H) [Li et al., 1993; Legay et al., 1993a]. An additional readthrough mRNA species retaining the intronic sequence I4 located immediately 3' to exon E4 was reported in rodent bone marrow and erythroleukemic cells [Li et al., 1993; Legay et al., 1993a] and in various tumor cells lines of human origin [Karpel et al., 1994].
The protein products of ACHE and BCHE differ in their tissue specificity. AChE is the major cholinesterase (ChE) in nervous system cells (20-fold more abundant than BuChE in the brain). In contrast, BuChE is the major blood ChE (3-fold more abundant than AChE). Since acetylcholine is produced mostly in the CNS, changes in AChE should be coupled to mental state.
Several experimental models have demonstrated morphogenic activities for AChE [Layer, 1995] and in particular involvement in Alzheimer's Disease (AD). Currently approved drugs for the treatment of Alzheimer's disease patients are designed to suppress the catalytic activity of the acetylcholine hydrolyzing enzyme acetylcholinesterase (acetylcholine acetyl hydrolase, EC 3.1.1.7, AChE) [Knapp et al., 1994]. This is aimed at augmentation of cholinergic neurotransmission, which is impaired in such patients due to a selective loss of cholinergic neurons. However, such inhibitors do not reduce the amount of AChE protein, and there are recent reports of actions of AChE, unrelated to its catalytic activity, in process extension [Small et al., 1995, Layer et al.; 1995, Jones et al., 1995; Darboux et al., 1996; Sternfeld et al., 1997] and amyloid fibril formation [Inestrosa et al., 1996].
The only currently approved drug for Alzheimer's Disease is Tacrine, a potent blocker of acetylcholinesterase activity. Tacrine offers limited palliative relief to 30-50% of mild-moderately affected Alzheimer's patients for up to 6 months [Knapp et al., 1994].
The positive, albeit partial, success of Tacrine attests to the potential value of improved anticholinesterase treatment of Alzheimer's Disease. However, anti-acetylcholinesterase therapies for Alzheimer's Disease require high doses of drug and produce side-effects associated with systemic cholinergic toxicity. Tacrine, for example, has been associated with liver damage and blood disorders in some patients. These considerations indicate the need to develop a new generation of anti-acetylcholinesterase drugs displaying increased target specificity, improved efficacy and reduced side effects.
Breakthroughs in molecular biology and the human genome project have opened previously unforeseen possibilities for targeted intervention with mammalian gene expression. These include permanent approaches such as transgenic overexpression or recombinant disruption of specific genes as well as novel approaches for transient suppression of gene function. Short synthetic antisense (AS) oligodeoxynucleotides (AS-ODN) designed to hybridize with specific sequences within a targeted mRNA belong to the latter class.
Many excellent reviews have covered the main aspects of antisense technology and its enormous therapeutic potential. The literature naturally progressed from chemical [Crooke, 1995] into cellular [Wagner, 1994] and therapeutic [Hanania, et al, 1995; Scanlon, et al, 1995] aspects of this rapidly developing technology. Within a relatively short time, ample information has accumulated about the in vitro use of AS-ODN in cultured primary cells and cell lines as well as for in vivo administration of such ODNs for suppressing specific processes and changing body functions in a transient manner. This wealth of accumulated experience now offers a novel way to analyze the antisense approach, namely, to compare its in vitro uses with its in vivo ones [Lev-Lehman et al, 1997]. Further, enough experience is now available in vitro and in vivo in animal models as shown in the Examples of the present application to predict human efficacy.
AS intervention in the expression of specific genes can be achieved by the use of synthetic AS-ODNs [for recent reports see Lefebvre-d'Hellencourt et al, 1995; Agrawal, 1996; Lev-Lehman et al, 1997]. AS-ODNs are short sequences of DNA (15-25 mer) designed to complement a target mRNA of interest and form an RNA:ODN duplex. This duplex formation can prevent processing, splicing, transport or translation of the relevant mRNA. Moreover, certain AS-ODNs can elicit cellular RNase H activity when hybridized with their target mRNA, resulting in mRNA degradation [Calabretta et al, 1996]. In that case, RNase H will cleave the RNA component of the duplex and can potentially release the AS-ODN to further hybridize with additional molecules of the target RNA. An additional mode of action results from the interaction of AS-ODNs with genomic DNA to form a triple helix which may be transcriptionally inactive. See FIG. 1 for a schematic representation of the modes of action of AS-ODN.
Phosphorothioate antisense oligonucleotides do not show significant toxicity and exhibit sufficient pharmacodynamic half-lives in animals [Agarwal et al., 1991, 1996]. Antisense induced loss-of-function phenotypes related with cellular development were shown for the glial fibrillary acidic protein (GFAP), implicated in astrocyte growth within astrocyte-neuron cocultures [Winstein et al., 1991], for the myelin-associated glycoprotein in Schwann cells, responsible for formation of the compact myelin sheath formation surrounding these cell [Owens and Bunge, 1991], for the microtubule-associated tau proteins implicated with the polarity of hippocampal neurons and their axon formation [Caceres and Kosik, 1990], for the .beta..sub.1 -integrin, important for neuronal migration along radial glial cells, and for the establishment of tectal plate formation in chick [Galileo et al., 1991] and for the N-myc protein, responsible for the maintenance of cellular heterogeneity in neuroectodermal cultures (ephithelial vs. neuroblastic cells, which differ in their colony forming abilities, tumorigenicity and adherence) [Rosolen et al., 1990; Whitesell et al, 1991]. Antisense oligonucleotide inhibition of basic fibroblast growth factor (bFgF), having mitogenic and angiogenic properties, suppressed 80% of growth in glioma cells [Morrison, 1991] in a saturable and specific manner. The antisense oligonucleotides were targeted against the initiation and splice sites in bFgFmRNA, they reduced activity of the resulting protein and sense oligomers remained inactive. In soft-agar cultures, antisense oligonucleotides reduced the size of glial colonies and induced appearance of larger cells within them [Morrison, 1992]. Being hydrophobic, antisense oligonucleotides interact well with phospholipid membranes [Akhter et al., 1991]. Following their interaction with the cellular plasma membrane, they are actively transported into living cells [Loke et al., 1989], in a saturable mechanism predicted to involve specific receptors [Yakubov et al., 1989].
AChE inhibitors such as tacrine also interact with serum BuChE as well, complicating individual variability regarding pharmacokinetics. Moreover, since they only interfere with enzymatic activity they would not necessarily prevent the non-cholinolytic action of ChEs. ChEs affect cell growth and/or cell adhesion also in the presence of tacrine and related drugs which may be the aspect associated with disease. This emphasizes the inherent advantage of the antisense approach for suppressing AChE protein production: such treatment will be sequence-specific, avoiding interference with BuChE production and exert their suppression activity on fully differentiated neurons. It can selectively prevent both catalytic and/or non-catalytic effects of AChE, unlike most chemical inhibitors, with a clear added value for suppressing the undesirable effects of AChE overexpression and only those.